EP0558747B1 - Immunadsorbens zur diagnose von epilepsie und der risikogruppe - Google Patents

Immunadsorbens zur diagnose von epilepsie und der risikogruppe Download PDF

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Publication number
EP0558747B1
EP0558747B1 EP91912818A EP91912818A EP0558747B1 EP 0558747 B1 EP0558747 B1 EP 0558747B1 EP 91912818 A EP91912818 A EP 91912818A EP 91912818 A EP91912818 A EP 91912818A EP 0558747 B1 EP0558747 B1 EP 0558747B1
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EP
European Patent Office
Prior art keywords
glutamate
membrane protein
immunosorbent
epilepsy
binding membrane
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP91912818A
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English (en)
French (fr)
Other versions
EP0558747A4 (en
EP0558747A1 (de
Inventor
Svetlana Alexandrovna Dambinova
Alexandr Izarovich Gorodinsky
Marina Nikolaevna Demina
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OBSCHESTVO S OGRANICHENNOI OTVETSTVENNOSTJU " DIES"
Original Assignee
OBSCHESTVO S OGRANICHENNOI OTVETSTVENNOSTJU " DIES"
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Priority to AT91912818T priority Critical patent/ATE161961T1/de
Publication of EP0558747A1 publication Critical patent/EP0558747A1/de
Publication of EP0558747A4 publication Critical patent/EP0558747A4/en
Application granted granted Critical
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2857—Seizure disorders; Epilepsy

Definitions

  • the present invention relates generally to medicine and more specifically to the immunosorbent for epilepsy and the risk group identification, finding application in the psychoneurological practice for the epilepsy diagnosis and the risk-group identification.
  • Epilepsy is a severe brain disease that affects about 1-4% of population.
  • diagnosis is carried out on the basis of patients's clinical investigation which includes electrophysiological and X-ray techniques as well as other instrumentations.
  • Such approach does not permit to evaluate unambiguously the brain excitatory pathways disturbances and related central nervous system disorders.
  • possible risk group identification is virtually excluded.
  • the biochemical analysis of the patient's biological fluids for neurotransmitters (glutamate, gamma-aminobutyric acid, serotonin, dopamine and others) or their metabolites is usually used.
  • neurotransmitters glutamate, gamma-aminobutyric acid, serotonin, dopamine and others
  • their metabolites are usually used.
  • such analysis does not provide the expected results, not allowing unambiguous evaluation of specific metabolic disturbances in the brain, predisposition (risk group) identification, and the diagnosis on the early stages of the disease.
  • the herein-proposed immunosorbent for epilepsy and the risk group identification is substantially novel and has not so far been described in literature.
  • said object is accomplished by the provision of a trypsin digest fragment of the glutamate-binding membrane protein from bovine brain tissue having a molecular weight of 5 to 14 kilodaltons, which is bound covalently or ionically to a support material, whereby the preservation of its immunogenic properties is ensured.
  • the support material is polystyrene, which is aminated and subsequently derivatized with glutaraldehyde, or nitrocellulose.
  • the proposed immunosorbent enables the rapid and effective diagnosis of the epilepsy in a substantial number of patients (up to 200 individuals) simultaneously. Additionally the proposed immunosorbent enables to carry out the prophylactic population screening for the risk group determination and the professional aptitude test with a view to evaluate risk groups among personnel expected to operate under stressing conditions.
  • a diagnosis "epilepsy” is made on the base of two- to four-fold excess of autoantibodies' concentration above control level. Response of the immunosorbent to the blood of patients with other nervous system diseases do not essentially differ from the control level.
  • the proposed immunosorbent was tested on the laboratory epilepsy model - Krushinsky-Molodkina rats with the inherent predisposition to audiogenic seizures. Normal Wistar rats were used as a control.
  • the rats were exposed to the acoustical treatment (96 DB), which caused the complete audiogenic seizure pattern.
  • the blood was sampled from the tail vein, frozen and stored at -60°C until use.
  • the data obtained provide the evidence of the relationship between the inheritable predisposition to audiogenic seizures and the blood level of anti-glutamate-binding membrane protein autoantibodies.
  • the autoantibodies detected in the blood of Krushinsky-Molodkina rats promote the audiogenic seizure which in turn cause further autoantibodies level elevation.
  • the proposed immunosorbent was tested in clinic on the blood samples of more than 1000 patients with epilepsy (650 patients), epileptiform syndrome of unknown etiology (187), Parkinson's disease (148) und other neurologic diseases, including schizophrenia (47), Alzheimer's disease (14), as well as 2150 healthy volunteers.
  • the patients' and healthy controls' serum samples were applied in the microtiter wells of the proposed immunosorbent which is the fragment of the glutamate-binding membrane protein from the mammalian brain having a molecular weight of 5 to 14 kilodaltons bound covalently to polystyrene, treated with glutaraldehyde.
  • the rabbit polyclonal antiserum against the glutamate-binding membrane protein (titer 1:36000), applied in the microtiter wells in 1:5000 dilution simultaneously with the patients' blood samples, was used.
  • the basal (zero) level was adjusted according to the analysis data of donor serum depleted by the immunoprecipitation with rat brain membrane suspension.
  • the increased level of autoantibodies to the glutamate-binding membrane protein was observed in the blood of patients with epilepsy and epileptiform syndrome of unknown etiology. In these cases the analysis revealed 89 - 90% of patients. The binding to the immunosorbent of the blood of patients with Parkinson's disease and other central nervous system disorders did not differ from the control level. Other antigens exhibited substantially less specificity.
  • the epileptic patients' recognition level by total synaptic membranes solubilizate was about 60% of the value obtained with the glutamate-binding membrane protein, and by the liver, heart and kidney membranes solubilizates - 32, 36 and 21 % respectively.
  • the purified opiate-binding membrane protein did not virtually bind the components of epileptic sera.
  • the proposed immunosorbent would enable the rapid and effective diagnosis of the epilepsy in a substantial number of patients (up to 200 individuals) simultaneously and to carry out the prophylactic population screening for the risk group determination and the professional aptitude test with a view to evaluate risk groups among personnel expected to operate under stressing conditions.
  • Clinical application of the proposed immunosorbent would allow to improve the patients' status monitoring and optimize the treatment process.
  • the proposed immunosorbent is produced as follows.
  • the fragment of the glutamate-binding membrane protein from the mammalian brain having a molecular weight of 5 to 14 kilodaltons is bound covalently or ionically to the support material which ensures the preservation of its immunogenic properties.
  • a support material polystyrene, chromatography paper, nitrocellulose and other materials can be applied.
  • the polystyrene microtiter plate is treated by nitration to form free nitro groups on the plate surface.
  • the nitro groups are reduced to aminogroups and activated by glutaraldehyde which serves as a linking factor.
  • glutaraldehyde which serves as a linking factor.
  • the activated plates are incubated with the previously isolated fragment for 16 hours at 4°C.
  • the non-bound active groups reduction by 0.1% (weight/volume) of sodium borohydride and washing the wells with 0.9% (weight/volume) of sodium chloride
  • the plate is incubated with a preservative, dried under vacuum and hermetically sealed. After the preservation microtiter plates with the linked immunogenic fragment of the glutamate-binding membrane protein are stored at 4°C.
  • the immunosorbent by binding the immunogenic fragment of the glutamate-binding membrane protein to nitrocellulose strips by the ionic interaction.
  • an aqueous solution containing the immunogenic fragment of the glutamate-binding membrane protein is applied to the strips and incubated 15 min. Unbound nitrocellulose active groups are blocked by 0.5% (weight/volume) serum albumin with 0.05% (volume/volume) detergent Tween 20. Dried strips are stored up to 1 year in dry place.
  • the glutamate-binding membrane protein is isolated from bovine cerebral cortex. Cortex portions (140 g) are homogenized in 500 ml of ice-cold 0.32 M sucrose solution containing phenylmethylsulfonyl fluoride (0.1 mM) and centrifuged at 800 g for 10 min. Supernatant is filtered through 4 layers of nylon gauze and centrifuged at 20000 g for 30 min. The resulting pellet is resuspended in 500 ml of ice-cold distilled water and centrifuged at 8000 g for 30 min. The procedure is repeated twice and the supernatant together with the pellet soft layer is centrifuged at 48000 g 30 min.
  • the resulting fraction (synaptic membranes) is suspended in 1% (weight/volume) solution of sodium deoxycholate in 30 mM Tris-HCl, pH 7.4 and stirred 1 hour at 4°C.
  • the solubilizate is cleared by centrifugation (100000 g for 1.5 hour) and mixed with glutamate-Sepharose 4B (Pharmacia, Sweden), previously suspended in 30 mM Tris-HCl (pH 7.4).
  • the protein binding to the sorbent lasts 16 hours at 4°C.
  • the glutamate-binding membrane protein output accounts for about 2 ⁇ g/g of brain tissue.
  • the immunogenic fragment of the glutamate-binding membrane protein 200 pg of the purified protein is incubated in 20 ml of trypsin solution ("Sigma", USA, type III, 1 mg/mg of glutamate-binding membrane protein) for 3 hours at 37°C. The reaction is terminated by the equivalent quantity of trypsin inhibitor ("Sigma", USA) and the reaction mixture is applied on the Ultrasphere C-18 column (4.6 x 250 mm) for high-performance liquid chromatography. The elution is run with the linear acetonitrile gradient (20 - 68%) at a speed of 1.0 ml/min.
  • the immunogenic fragment of the glutamate-binding membrane protein is characterized biochemically, as the electrophoretically homogeneous polypeptide having a molecular weight of 5 to 14 kilodaltons, and immunologically according to its binding to monoclonal and polyclonal antibodies against the glutamate-binding membrane protein and negative reaction with control antisera against other proteins (interferon and alkaline phosphatase).
  • the immunogenic fragment of the glutamate-binding membrane protein (0.1 ⁇ g in 0.1 ml) is added in each well.
  • the protein binding to the plate continues for 16 hours at 4°C.
  • Aldehyde groups not involved in the reaction are reduced with fresh-prepared sodium borohydride 0,1% (weight/ volume) solution for 30 min at room temperature.
  • the wells are washed with the same buffer and loaded with the preservative: mixture of 5% (weight/volume) sucrose and 1% (weight/volume) bovine serum albumin. After the incubation for 1.5 hours at room temperature the plate is vacuum-dried.
  • the immunosorbent is produced which is in fact the fragment of the glutamate-binding membrane protein having a molecular weight of 5 to 14 kilodaltons bound covalently to the polystyrene microtiter plate.
  • Produced immunosorbent is stored at 4°C for 12 months.
  • the immunogenic fragment of the glutamate-binding membrane protein is isolated after trypsin incubation and chromatography as described in Example 1.
  • the nitrocellulose sheet (80 x 120 mm 2 ) is inserted in the apparatus for dot-blotting and the wells are filled with the purified glutamate-binding membrane protein-solution. After incubation for 15 min the excess solution is removed by vacuum-filtration, and the nitrocellulose sheets are transferred in an aqueous medium containing 0.5% (weight/volume) Tween 20. After incubation at room temperature for 1 hour the sheets are dried under vacuum.
  • the immunosorbent produced is in fact the immunogenic fragment of the glutamate-binding membrane protein having a molecular weight of 5 to 14 kilodaltons bound ionically to the nitrocellulose. It is stored in a dry place at room temperature for 12 months.
  • the proposed immunosorbent can be used in the medical practice for the epilepsy diagnosis and the risk group identification.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Rheumatology (AREA)
  • Rehabilitation Therapy (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Claims (2)

  1. Immunosorbens für die Erkennung von Epilepsie und eine Prädisposition für Epilepsie, dadurch gekennzeichnet, daß es das Trypsinverdauungsfragment des glutamatbindenden Membranproteins aus Rinderhirngewebe mit einem Molekulargewicht von 5 bis 14 Kilodalton darstellt, das kovalent oder ionisch an einen Trägerstoff gebunden ist, wodurch die Aufrechterhaltung seiner immunogenen Eigenschaften gewährleistet wird.
  2. Immunosorbens nach Anspruch 1, dadurch gekennzeichnet, daß der Trägerstoff aminiertes und nachfolgend mit Glutaraldehyd deriviertes Polystyrol oder Nitrozellulose ist.
EP91912818A 1991-06-26 1991-06-26 Immunadsorbens zur diagnose von epilepsie und der risikogruppe Expired - Lifetime EP0558747B1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT91912818T ATE161961T1 (de) 1991-06-26 1991-06-26 Immunadsorbens zur diagnose von epilepsie und der risikogruppe

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/SU1991/000123 WO1993000586A1 (en) 1991-06-26 1991-06-26 Immunosorbent for diagnosis of epilepsy and group of risk

Publications (3)

Publication Number Publication Date
EP0558747A1 EP0558747A1 (de) 1993-09-08
EP0558747A4 EP0558747A4 (en) 1994-05-18
EP0558747B1 true EP0558747B1 (de) 1998-01-07

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EP91912818A Expired - Lifetime EP0558747B1 (de) 1991-06-26 1991-06-26 Immunadsorbens zur diagnose von epilepsie und der risikogruppe

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EP (1) EP0558747B1 (de)
DE (1) DE69128642T2 (de)
WO (1) WO1993000586A1 (de)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5529898A (en) * 1993-08-19 1996-06-25 Duke University Methods of detecting disorders of the central nervous system by detecting autoantibodies which specifically bind ionotropic glutamate receptors
EA200600920A1 (ru) 2003-11-06 2007-04-27 Грэйс Лэборетериз, Инк. Иммуносорбентные исследования крови для оценки пароксизмальных церебральных разрядов

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3346795A1 (de) * 1983-12-23 1985-07-04 E. Prof. Dr. 3550 Marburg Geyer Enzymimmunoassay und entsprechendes diagnostikum
JPS62132172A (ja) * 1985-12-04 1987-06-15 Shionogi & Co Ltd 固相化抗体およびその製造方法
US4962023A (en) * 1987-06-22 1990-10-09 Louisiana State University, Agricultural And Mechanical College Single incubation immuno sorbent assay method using particle labels to detect test antigen specific antibodies in presence of other antibodies

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Publication number Publication date
EP0558747A4 (en) 1994-05-18
DE69128642T2 (de) 1998-08-06
EP0558747A1 (de) 1993-09-08
WO1993000586A1 (en) 1993-01-07
DE69128642D1 (de) 1998-02-12

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